Compositions and methods for controlling food intake of animal

ABSTRACT

A composition comprising an active agent that inhibits acyl-coenzyme A:cholesterol acyltransferase (ACAT) is provided. With the composition, food intake can be suppressed, and/or body weight can be reduced, and/or metabolic disorders can be prevented and/or treated.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. application Ser.No. 17/343,589 filed on Jun. 9, 2021, which is a continuationapplication of U.S. application Ser. No. 16/461,597 filed on May 16,2019, which is a U.S. national stage entry of International ApplicationNo. PCT/US17/061893 filed on Nov. 16, 2017, which claims the benefits ofU.S. Provisional application 62/422,722, filed on Nov. 16, 2016. Theapplications are incorporated herein by reference.

REFERENCE TO ELECTRONIC SEQUENCE LISTING

The application contains a Sequence Listing which has been submittedelectronically in .XML format and is hereby incorporated by reference inits entirety. Said .XML copy, created on Mar. 28, 2023, is named“PUR-P30001C1C2-US.xml” and is 9,744 bytes in size. The sequence listingcontained in this .XML file is part of the specification and is herebyincorporated by reference herein in its entirety.

TECHNICAL FIELD

The present invention relates to methods and compositions forsuppressing food intake, suppressing body weight, and treating orpreventing metabolic diseases.

BACKGROUND

Obesity is an increasingly prevalent disease that has been shown tocontribute to a variety of life-threatening medical conditions. Suchmedical conditions include cardiovascular diseases and metabolicdiseases. Current efforts to treat obesity-related diseases are focusedon developing drugs or inhibitors of enzymes, transcription factors,and/or signaling proteins involved in neutral lipid (triglyceride)synthesis. However, given that there is still an increasing prevalenceof obesity, especially in the United States, there remains a significantneed for the identification of additional drug targets for thedevelopment of new, safe and effective anti-obesity drugs.

Hyperplasia and hypertrophy are the two major contributors to fat massexpansion during obesity development. Lipid droplet (LD) formation andenlargement are the characteristics of hyperplasia and hypertrophy,respectively. However, the molecular mechanisms regulating LDdevelopment in adipocytes have not been fully elucidated (Farese andWalther 2009).

Adipose tissue is also a cholesterol storage organ, in which themajority of the adipocyte cholesterol is found in LDs as freecholesterol (FC) and cholesterol ester (CE) (Farkas, et al. 1973).Several studies also reported a positive correlation betweenintracellular cholesterol level and TG content (Farkas, et al. 1973,LeLay, et al. 2001, Kovanen, et al. 1975, Schreibman, et al. 1975).Adipose cholesterol content in adipocytes appears to be associated withhuman obesity as obese humans are reported to store 33-50% of bodycholesterol in adipose tissue, while lean ones have about 25%(Schreibman, et al. 1975). LDs found in adipocytes are composed of aneutral lipid core and a phospholipid monolayer (Thiam, et al. 2013).The neutral lipid core contains mainly TG and DG, with some CE and otherlipid soluble compounds. The phospholipid monolayer mainly containsphospholipid, cholesterol and proteins associated with LD function. Itis known that more than 2000 proteins are increased or decreased ≥2folds during adipogenesis (Welsh, et al. 2004), including proteinsassociated with lipid synthesis. It is also known that the cholesterolcontent increases as LD grows, and restricting the cholesterol contentcan block the development of LD (Dagher, et al. 2003).

Acyl CoA:cholesterol acyl transferases (ACATs) catalyze the formation ofCE from long chain fatty acids and cholesterol in the presence of ATPand coenzyme A (Mukherjee, et al. 1958, Chang, et al. 1988, Zhang, etal. 2003). ACAT1 is ubiquitously expressed in different tissues tomaintain cholesterol homeostasis, while ACAT2 is known to be mainlyexpressed in the liver and intestine for CE supply, respectively(Tomoda, et al. 2007). Both ACAT1 and ACAT2 are considered to be drugtargets for the treatments of not only atherosclerosis (Yagyu, et al.2000, Fazio, et al. 2001) but also cancer (Yue, et al. 2014) andAlzheimer's diseases (Shibuya, et al. 2015, Bhattacharyya, et al. 2010).Moreover, this invention demonstrates that ACATs are required for LDformation and increasing intracellular cholesterol content duringadipogenesis in vitro. Therefore, ACAT may play a critical role in thenetwork of LD development. Since cholesterol homeostasis impactsadipocyte function (Yu, et al. 2010), ACAT inhibition may impact variousbiological processes besides lipogenesis.

Diabetes is the 7^(th) leading cause of death affecting more than 10% ofadults and 26% of seniors of the U.S. population, largely due to itsrelated health complications, including hypertension, heart attack,stroke, kidney diseases and eye problems. The total cost of diagnoseddiabetes in the U.S. is approximately $245 billion and around $176billion is spent for direct medical costs. Type 2 diabetes (T2D) ischaracterized by “insulin resistance” as the body doesn't properlyhandle blood glucose when insulin is present. About 90-95% of all NorthAmerican cases of diabetes are T2D. T2D and obesity are closelyinter-connected as more than 85% of patients with T2D are also obese.Indeed, obesity has been shown to contribute to a variety oflife-threatening medical conditions such as T2D and its related healthcomplications.

Currently, approximately 10 of the best-selling anti-diabetes drugscomprise approximately $28.6 billion globally. Many of the availableanti-diabetes drugs (e.g., rosiglitazone, pioglitazone, sitagliptin,saxagliptin, liragliptin, exenatide, liraglutide and albiglutide) areassociated with an undesirable increase or little effect on adipose massand weight gain, which compromise the overall efficacy of diabetestherapy as well as the quality of life for the patients. Given the factsthat the majority of T2D patients is already overweight and obese,physicians are often recommendation to elect T2D drugs with favorableadditional effects on weight loss.

Combined effect of a PPARα/γ dual agonist and ACAT inhibitors on thetreatment of hyperglycemia, lipid disorders, and obesity in a patienthaving T2D was reported (e.g., WO 2003/088962). However, no evidence ofdirect effects of ACAT inhibitor alone on body weight, food intake andinsulin resistance was presented.

Various methods for treating lipid disorders or hyperlipidemia andsuppressing obesity by administering ACAT2 inhibitors andACAT1-selective inhibitors orally or parenterally have been proposed, asdisclosed in, e.g., WO 2009/081957, KR1020030011474, U.S. ApplicationPublication No. 2011/0184173, EP2228376, and WO2015065595. Nowhere,however, do these disclosures indicate that administration of ACATinhibitors were associated with suppression of food intake andits-related body weight loss and insulin sensitivity.

This BACKGROUND section introduces aspects that may help facilitate abetter understanding of the disclosure. Accordingly, it should be readin this light and it should not be understood as admissions about whatis or is not prior art. The references cited herein are incorporatedherein by reference.

SUMMARY

In one aspect, the present invention provides a composition forsuppressing food intake, reducing body weight, and/orpreventing/treating metabolic disorders. The composition comprises aneffective amount of an active agent that can inhibit acyl-coenzymeA:cholesterol acyltransferase (ACAT).

In some embodiments, the active agent can inhibit ACAT1, ACAT2, or both.In some cases, the degree that the active agent inhibits ACAT1 may besimilar to or less than the degree that the active agent inhibits ACAT2.

In some embodiments, the composition may comprise avasimibe as theactive agent. The composition may comprise CI-976 as the active agent.The composition may comprise both avasimibe and CI-976 as the activeagents.

In another aspect, the present invention provides a method forsuppressing food intake of a subject. The method comprises administeringto the subject the above-described composition.

In some embodiments, the effective amount may be in the range of about0.01 mg/kg/day to about 200 mg/kg/day.

In some embodiments, the composition may be administered parenterally.For example, the composition may be administered subcutaneously,intravenously, or intraperitoneally.

In some embodiments, the method can reduce body weight. The method canprevent or treat metabolic disorders.

In some embodiment, the high fat diet fed subject administered with theactive agent has decreased level of free cholesterol, CE and TG comparedto a subject having high fat diet without the active agentadministration.

In another aspect the present invention provides an shRNA sequence toknockdown ACAT1 comprising SEQ ID NO:5.

In another aspect the present invention provides an shRNA sequence toknockdown ACAT2 comprising SEQ ID NO:6.

In yet another aspect the present invention provides a method ofinhibiting synthesis of fatty acids and TGs in adipocytes. The methodcomprises introducing an shRNA sequence operably integrated to alentivirus vector to the adipocytes, wherein the shRNA sequence isselected from the group consisting of SEQ ID NO:5 and SEQ ID NO:6.

Each embodiment disclosed herein is contemplated as being applicable toeach of the other disclosed embodiments. Thus, all combinations of thevarious elements described herein are within the scope of the invention.Other aspects are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the relative mRNA expression of ACAT1 andACAT2 in preadipocytes (DO), immature adipocytes (D2) and matureadipocytes (D6).

FIG. 2 is a bar graph showing the transcript levels of ACAT1, ACAT2,leptin, UCP-1 and FAS relative to RPL27 in white adipose tissue (WAT),brown adipose tissue (BAT) and the liver of lean mice and high fatdiet-induced obese mice.

FIG. 3A is an image showing lipid accumulation in preadipocytes andmature adipocytes differentiated in the presence of variousconcentrations of avasimibe, visualized by Oil Red O staining. FIG. 3Bis a bar graph showing the quantified Oil Red O stained-lipidaccumulation shown in FIG. 3A.

FIG. 4 is a Coherent Anti-Stokes Raman Scattering (CARS) microscopyimage showing the suppressed lipid accumulation by avasimibe in matureadipocytes.

FIG. 5A is an image showing lipid accumulation in preadipocytes andmature adipocytes differentiated in the presence of variousconcentrations of CI-976, visualized by Oil Red O staining. FIG. 5B is abar graph showing the quantified Oil Red O stained-lipid accumulationshown in FIG. 5A.

FIG. 6 is a bar graph showing that avasimibe suppresses mRNA levels ofgenes involved in lipid synthesis in adipocytes.

FIG. 7 is a Western blotting image showing that avasimibe increases thelevel of the 125 kDa inactive precursor form of SREBP1 in adipocytes.

FIG. 8 is a stimulated Raman scattering (SRS) microscopy image showingthe suppressive role of avasimibe in de novo fatty acid synthesis inadipocytes.

FIG. 9 is a bar graph showing that avasimibe completely inhibitscholesterol ester (CE) accumulation and suppresses the cholesterol levelin adipocytes during adipogenesis.

FIG. 10 is a bar graph showing that avasimibe suppresses the relativemRNA levels of SR-BI and CD-36 in adipocytes.

FIG. 11 is an image of the25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol(25-NBD cholesterol)-based ACAT assay showing that avasimibe suppressesthe ACAT activity and thereby suppresses accumulation offluorescence-labeled CE in adipocytes.

FIG. 12 is a bar graph showing the efficiency of the ACAT1 shRNA toknockdown the ACAT1 expression without altering the ACAT2 expressionlevel in adipocytes.

FIG. 13A is an image showing the effect of ACAT1 knockdown on lipidaccumulation in adipocytes, visualized by Oil Red O staining. FIG. 13Bis a bar graph showing the quantified Oil Red O stained-lipidaccumulation shown in FIG. 13A.

FIG. 14 is a bar graph showing that ACAT1 knockdown reduces mRNA levelsof genes involved in lipid synthesis in adipocytes.

FIG. 15A is an image showing the effect of ACAT2 knockdown on lipidaccumulation in adipocytes, visualized by Oil Red O staining. FIG. 15Bis a bar graph showing the quantified Oil Red O stained-lipidaccumulation shown in FIG. 15A.

FIG. 16 is a bar graph showing that ACAT2 knockdown reduces mRNA levelsof genes involved in lipid synthesis in adipocytes.

FIG. 17A is a graph showing the progressive loss of body weight byavasimibe in a high-fat diet-induced obesity mouse model. FIG. 17B is agraph representing the percentage of the body weight loss by avasimibein a high-fat diet-induced obesity mouse model.

FIG. 18A is a bar graph showing the loss of whole body fat mass byavasimibe in a high-fat diet-induced obesity mouse model. FIG. 18B is abar graph showing the loss of white adipose tissues (Ing: inguinal, Epi:epididymal) weight by avasimibe in a high-fat diet-induced obesity mousemodel, compared with non-adipose tissues.

FIG. 19 is a graph representing the results of the serum alaninetransaminase assay showing that avasimibe does not exhibit noticeableliver toxicity.

FIG. 20A is a bar graph showing the suppressed food intake by avasimibein a high-fat diet-induced obesity mouse model. FIG. 20B and FIG. 20Care bar graphs showing the suppressed energy expenditure and thesuppressed respiratory exchange ratio by avasimibe in a high-fatdiet-induced obesity mouse model, encompassing the dark and the lightphases.

FIG. 21A and FIG. 21B are graphs showing the suppressed blood glucoselevels and the suppressed insulin levels by avasimibe in a high-fatdiet-induced obesity mouse model. FIG. 21C is a graph showing theimproved glucose tolerance by avasimibe in a high-fat diet-inducedobesity mouse model. FIG. 21D is a bar graph showing the improvedhomeostatic model assessment of insulin resistance (HOMA-IR) value byavasimibe in a high-fat diet-induced obesity mouse model.

FIG. 22A, FIG. 22B and FIG. 22C are graphs showing the suppressed serumfree cholesterol level, the suppressed serum cholesteryl ester level andthe suppressed serum triglyceride (TG) level by avasimibe in a high-fatdiet-induced obesity mouse model. FIG. 22D is a graph showing thesuppressed serum leptin level by avasimibe in a high-fat diet-inducedobesity mouse model.

FIG. 23A is a graph showing the progressive loss of body weight byavasimibe and by pair-feeding in a high-fat diet-induced obesity mousemodel. FIG. 23B, FIG. 23C and FIG. 23D are bar graphs showing thesuppressed food intake, the suppressed fecal excretion and thesuppressed fecal energy excretion by avasimibe and by pair-feeding in ahigh-fat diet-induced obesity mouse model. FIG. 23E is a graph showingthat pair-feeding suppresses the blood glucose level in a high-fatdiet-induced obesity mouse model, but not as effectively as avasimibedoes.

FIG. 24 is a pie chart showing the number of proteins influenced byavasimibe in adipocytes identified through LC MS/MS.

FIG. 25 is a list of biological processes to which proteins upregulatedby avasimibe in adipocytes relate.

FIG. 26 and FIG. 27 are lists of biological processes to which proteinsdownregulated by avasimibe in adipocytes relate.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended.

Definitions

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Thus, for example, reference to “a polypeptide” includesmixtures of polypeptides, reference to “a pharmaceutical carrier”includes mixtures of two or more such carriers, and the like.

As defined herein, the terms “about” or “approximately” mean within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 1 or more than 1 standarddeviation, per the practice in the art. Alternatively, “about” can meana range of up to 20%, up to 10%, up to 5%, or up to 1% of a given valueor range. Alternatively, particularly with respect to biological systemsor processes, the term can mean within an order of magnitude within5-fold, and also within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As defined herein, the terms “activation”, “activate”, “activating” andthe like in reference to a protein-activator (e.g. agonist) interactionmean positively affecting (e.g. increasing) the activity or function ofthe protein relative to the activity or function of the protein in theabsence of the activator (e.g. compound described herein). Thus,activation may include, at least in part, partially or totallyincreasing stimulation, increasing or enabling activation, oractivating, sensitizing, or up-regulating signal transduction orenzymatic activity or the amount of a harmful mediator/substancedecreased in a disease. Activation may include, at least in part,partially or totally increasing stimulation, increasing or enablingactivation, or activating, sensitizing, or up-regulating signaltransduction or enzymatic activity or the amount of a harmfulmediator/substance.

As defined herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intracranial, intranasal, intraocular, intracardiac, intravitreal,intracerebral, intraosseous, intraarterial, intraarticular, intradermal,transdermal, transmucosal, sublingual, enteral, sublabial, insufflation,inhaled or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc. By“coadminister” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional therapies (e.g. anti-obesityagent). The compound can be administered alone or can be coadministeredto the patient. Co-administration is meant to include simultaneous orsequential administration of the compound individually or in combination(more than one compound or agent). In some embodiments, a firstcomposition comprising a first ACAT inhibitor (e.g., (e.g., avasimibe)as the active agent and a second composition comprising a second ACATinhibitor (e.g., CI-976) as the active agent can be co-administered.

The preparations can also be combined, when desired, with other activesubstances (e.g. to reduce metabolic degradation, to increasedegradation of a prodrug and release of the drug, detectable agent). Thecompositions can be delivered transdermally, by a topical route,formulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.Oral preparations include tablets, pills, powder, dragees, capsules,liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc.,suitable for ingestion by the patient. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. Liquid form preparations include solutions,suspensions, and emulsions, for example, water or water/propylene glycolsolutions. The compositions may additionally include components toprovide sustained release and/or comfort. Such components include highmolecular weight, anionic mucomimetic polymers, gelling polysaccharidesand finely-divided drug carrier substrates. These components arediscussed in greater detail in U.S. Pat. Nos. 4,911,920; 5,403,841;5,212,162; and 4,861,760. The entire contents of these patents areincorporated herein by reference in their entirety for all purposes. Thecompositions can also be delivered as microspheres for slow release inthe body. For example, microspheres can be administered via intradermalinjection of drug-containing microspheres, which slowly releasesubcutaneously (see Rao, J. Biomater Sci. Polym. Ed. 7:623-645, 1995; asbiodegradable and injectable gel formulations (see, e.g., Gao Pharm.Res. 12:857-863, 1995); or, as microspheres for oral administration(see, e.g., Eyles, J. Pharm. Pharmacol. 49:669-674, 1997). In anotherembodiment, the formulations of the compositions can be delivered by theuse of liposomes which fuse with the cellular membrane or areendocytosed, i.e., by employing receptor ligands attached to theliposome, that bind to surface membrane protein receptors of the cellresulting in endocytosis. By using liposomes, particularly where theliposome surface carries receptor ligands specific for target cells, orare otherwise preferentially directed to a specific organ, one can focusthe delivery of the compositions into the target cells in vivo. (See,e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996; Chonn, Curr.Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp. Pharm.46:1576-1587, 1989). The compositions can also be delivered asnanoparticles.

As used herein, the term “agent” is meant to encompass any molecule,chemical entity, composition, drug, therapeutic agent, chemotherapeuticagent, or biological agent capable of preventing, ameliorating, ortreating a disease or other medical condition. The term includes smallmolecule compounds, antisense oligonucleotides, siRNA reagents,antibodies, antibody fragments bearing epitope recognition sites,peptoids, aptamers, enzymes, peptides organic or inorganic molecules,natural or synthetic compounds and the like. An agent can be assayed inaccordance with the methods of the invention at any stage duringclinical trials, during pre-trial testing, or following FDA-approval.

As defined herein, the terms “alter”, “altering” “alteration” and thelike are meant a change (increase or decrease) in the expression levelsor activity of a gene or polypeptide as detected by standard art knownmethods such as those described herein.

As defined herein, the term “ameliorate” is meant decrease, suppress,attenuate, diminish, arrest, or stabilize the development or progressionof a disease.

As defined herein, the terms “associated” or “associated with” in thecontext of a substance or substance activity or function associated witha disease (e.g. obesity) mean that the disease is caused by (in whole orin part), or a symptom of the disease is caused by (in whole or in part)the substance or substance activity or function. As used herein, what isdescribed as being associated with a disease, if a causative agent,could be a target for treatment of the disease. For example, a diseaseassociated with weight gain such as obesity may be treated with an agent(e.g. compound as described herein) effective for decreasing weightgain.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, in reference to defined or described elements of anitem, composition, apparatus, method, process, system, etc., are meantto be inclusive or open ended, permitting additional elements, therebyindicating that the defined or described item, composition, apparatus,method, process, system, etc. includes those specified elements—or, asappropriate, equivalents thereof—and that other elements can be includedand still fall within the scope/definition of the defined item,composition, apparatus, method, process, system, etc. The terms“consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments. Furthermore, to the extent that the terms “including,”“includes,” “having,” “has,” “with,” or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

As defined herein, the terms “control” or “standard control” are used inaccordance with its plain ordinary meaning and refers to a sample thatserves as a reference, usually a known reference, for comparison to atest sample. For example, a test sample can be taken from a patientsuspected of having a given disease (e.g., obesity or diabetes) andcompared to samples from a known obesity or diabetes patient, or a knownnormal (e.g., non-disease) individual. A control can also represent anaverage value gathered from a population of similar individuals, e.g.,obesity or diabetes patients or healthy individuals with a similarmedical background, same age, weight, etc. A control can also beobtained from the same individual, e.g., from an earlier-obtainedsample, prior to disease, or prior to treatment. One of skill willrecognize that controls can be designed for assessment of any number ofparameters, and one of skill will understand which controls are valuablein a given situation and be able to analyze data based on comparisons tocontrol values. Controls are also valuable for determining thesignificance of data. For example, if values for a given parameter arewidely variant in controls, variation in test samples will not beconsidered as significant.

As defined herein, the terms “disease” or “condition” refer to a stateof being or health status of a patient or subject capable of beingtreated with a compound, pharmaceutical composition, or method providedherein. In some embodiments, the disease is a disease having an increasein body weight. In some embodiments, the disease is obesity. Obesity maybe the primary cause of the disease and/or disease to be treated or mayalso by a result of the primary disease and/or disorder. In someembodiments, the disease is a metabolic disease, such as diabetes.

The terms “metabolic disease,” “metabolic disorder” and “metabolicsyndrome” are used interchangeably to refer to any condition in whichthere is a defect of metabolism, typically due to a genetic defect.Non-limiting examples of metabolic processes that can be impactedinclude carbohydrate, protein, and/or fat metabolic pathways in food torelease energy, transformation of excess nitrogen into waste productsexcreted in urine and the breaking down or converting chemicals intoother substances and transporting them inside cells.

As used herein, the term “effective amount” refers to an amountsufficient for a compound to accomplish a stated purpose relative to theabsence of the compound (e.g. achieve the effect for which it isadministered, treat a disease, reduce enzyme activity, increase enzymeactivity, reduce signaling pathway, reduce one or more symptoms of adisease or condition). An example of an “effective amount” is an amountsufficient to contribute to the treatment, prevention, or reduction of asymptom or symptoms of a disease, which could also be referred to as a“therapeutically effective amount.”

As defined herein, the term “in combination” in the context of theadministration of a therapy to a subject refers to the use of more thanone therapy (e.g., prophylactic and/or therapeutic). The use of the term“in combination” does not restrict the order in which the therapies,concomitantly with, or subsequent to the administration of a secondtherapy to a subject which had, has, or is susceptible to obesity ordiabetes. In some embodiments, the therapies are administered to asubject in a sequence and within a time interval such that the therapiescan act together. In some embodiments, the therapies are administered toa subject in a sequence and within a time interval such that theyprovide an increased benefit than if they were administered otherwise.Any additional therapy can be administered in any order with the otheradditional therapy.

As defined herein, the terms “inhibition,” “inhibit,” “inhibiting,” andthe like in reference to a protein-inhibitor (e.g. antagonist)interaction mean negatively affecting (e.g. decreasing) the level ofactivity or function of the protein relative to the level of activity orfunction of the protein in the absence of the inhibitor. In someembodiments, inhibition refers to reduction of a disease or symptoms ofdisease. Thus, inhibition may include, at least in part, partially ortotally blocking stimulation, decreasing, preventing, or delayingactivation, or inactivating, desensitizing, or down-regulating signaltransduction or enzymatic activity or the amount of a protein.

As defined herein, the terms “modulate,” “modulating,” and the likerefer to increasing or decreasing the level of a target molecule or thefunction of a target molecule.

As used herein, the term “or” is generally employed in its senseincluding “and/or” unless the content clearly dictates otherwise.

As defined herein, the terms “pharmaceutically acceptable excipient” and“pharmaceutically acceptable carrier” refer to a substance that aids theadministration of an active agent to and absorption by a subject and canbe included in the compositions of the present invention without causinga significant adverse toxicological effect on the patient. Non limitingexamples of pharmaceutically acceptable excipients include water, NaCl,normal saline solutions, lactated Ringer's, normal sucrose, normalglucose, binders, fillers, disintegrants, lubricants, coatings,sweeteners, flavors, salt solutions (such as Ringer's solution),alcohols, oils, gelatins, carbohydrates such as lactose, amylose orstarch, fatty acid esters, hydroxymethycellulose, polyvinyl pyrrolidine,and colors, and the like. Such preparations can be sterilized and, ifdesired, mixed with auxiliary agents such as lubricants, preservatives,stabilizers, wetting agents, emulsifiers, salts for influencing osmoticpressure, buffers, coloring, and/or aromatic substances and the likethat do not deleteriously react with the compounds of the invention. Oneof skill in the art will recognize that other pharmaceutical excipientsare useful in the present invention.

As defined herein, the terms “prevent,” “preventing,” “prevention,”“prophylactic treatment” and the like refer to reducing the probabilityof developing a disease or condition in a subject, who does not have,but is at risk of or susceptible to developing a disease or condition.

As defined herein, the term “prophylactically effective amount” of adrug is an amount of a drug that, when administered to a subject, willhave the intended prophylactic effect, e.g., preventing or delaying theonset (or reoccurrence) of an injury, disease, pathology or condition,or reducing the likelihood of the onset (or reoccurrence) of an injury,disease, pathology, or condition, or their symptoms. The fullprophylactic effect does not necessarily occur by administration of onedose, and it may occur only after administration of a series of doses.Thus, a prophylactically effective amount may be administered in one ormore administrations. An “activity decreasing amount,” as used herein,refers to an amount of antagonist required to decrease the activity ofan enzyme relative to the absence of the antagonist. The exact amountswill depend on the purpose of the treatment, and will be ascertainableby one skilled in the art using known techniques (see, e.g., Lieberman,Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Scienceand Technology of Pharmaceutical Compounding (1999); Pickar, DosageCalculations (1999); and Remington: The Science and Practice ofPharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams &Wilkins).

As defined herein, the terms “subject”, “patient”, “individual” and thelike refer to a living organism who suffers from or is susceptible to adisease or condition that can be treated by administration of a compoundor pharmaceutical composition as provided herein. Non-limiting examplesinclude humans, vertebrates, other mammals, bovines, birds, rats, mice,dogs, cats, horses, primate, fowls, pigs, apes, monkeys, goats, camels,sheep, cows, deer, and other non-mammalian animals. In some embodiments,the subject is a companion animal, such as a dog or a cat. In someembodiments, a patient is human. In some embodiments, the patient ispre-obese, obese or morbidly obese. In some embodiments, the patient isnot pre-obese, obese, or morbidly obese, but was formerly pre-obese,obese, or morbidly obese. In some embodiments, the patient wishes tolose weight or have a decreased appetite. Alternatively or in addition,a patient has an obesity-related disease or disorder. These examples arenot limiting. The terms “subject,” “patient,” “individual,” and the likeas used herein are not intended to be limiting and can be generallyinterchanged. That is, an individual described as a “patient” does notnecessarily have a given disease or is not necessarily under the care ofa medical professional, but it may be merely seeking or wish to havetreatment in the absence of medical advice (such as self-treatment). Inaccordance with the methods described herein, a “subject in need of” isa subject with overweight or obesity or related disease or T2D, or asubject having an increased risk of developing overweight or obesity T2Dor related disease.

As defined herein, the terms “suppression”, “suppress”, “suppressing”and the like, or the terms “reduction”, “reduce” or “reducing” and thelike of a symptom or symptoms (and grammatical equivalents of thisphrase) mean decreasing of the severity or frequency of the symptom(s),or elimination of the symptom(s).

As used herein, the terms “therapies” and “therapy” can refer to anyprotocol(s), method(s), composition(s), formulation(s), and/or agent(s)that can be used in the prevention or treatment of a disease or symptomassociated therewith. In certain embodiments, the terms “therapies” and“therapy” refer to biological therapy, supportive therapy, and/or othertherapies useful in treatment or prevention of a disease or symptomassociated therewith known to one of skill in the art.

As defined herein, the terms “treating” or “treatment” refer to anyindicia of success in the treatment or amelioration of an injury,disease, pathology or condition, including any objective or subjectiveparameter such as abatement; remission; diminishing of symptoms ormaking the injury, pathology or condition more tolerable to the patient;slowing in the rate of degeneration or decline; making the final pointof degeneration less debilitating; improving a patient's physical ormental well-being. The treatment or amelioration of symptoms can bebased on objective or subjective parameters; including the results of aphysical examination, neuropsychiatric exams, and/or a psychiatricevaluation. For example, certain methods herein treat diseasesassociated with weight gain such as obesity.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Obesity

Obesity is characterized by excessive WAT, which is attributed toincreased adipocyte number (hyperplasia) and lipid storage(hypertrophy). Adipocyte hyperplasia and hypertrophy are governed by anincrease in adipogenesis and LD accumulation, respectively. Therefore,regulating adipogenesis is an effective strategy to control obesity andits associated diseases, such as atherosclerosis and othercardiovascular diseases (CVDs).

The term “obese”, as used herein, refers to a patient having a body massindex of greater than 30 kg/m². “Overweight” and “pre-obese”, as usedherein, refer to patients having a body mass index of greater than 25kg/m². “Morbidly obese”, as used herein, refers to a patient having aBMI of greater than 40 mg/m², a BMI of greater than 35 kg/m² incombination with one or more co-morbidities, a BMI of greater than 30kg/m² in combination with uncontrollable diabetes, or combinationsthereof.

The term “obesity-related diseases,” as used herein, comprises obesity,pre-obesity, morbid obesity, Prader-Willi Syndrome, Pro-opiomelanocortin(POMC) deficiency obesity, LepR deficiency obesity, POMC heterozygousdeficiency obesity, and POMC epigenetic disorders, Alström syndrome,Hypothalamic Injury Associated Obesity, Non-alcoholic steatohepatitis,hyperlipidemia, hypertension, diabetes, lipodystrophy, fatty liver,Bardet-Biedl Syndrome, Cohen Syndrome, cardiovascular disease,arthritis, stroke, metabolic syndrome and MOMO (Macrosomia ObesityMacrocephaly Ocular abnormalities) Syndrome.

The term “anti-obesity agent” refers to the property of a substance ortreatment that reduces weight gain and promotes weight loss. Examples ofanti-obesity agents would be Sibutramine, Phentermine, Mazindol,Diethylpropion, Leptin, Orlistat, Beta-3 agonists, and Rimonabant.

In one aspect, the present invention provides methods of suppressingfood intake of a subject, comprising: administering an effective amountof an agent inhibiting ACAT to the subject. In some embodiments, theagent inhibits ACAT1 and/or ACAT2. In some embodiments, the degree thatthe active agent inhibits ACAT1 may be similar to or less than thedegree that the active agent inhibits ACAT 2. In some embodiments, theagent is selected from the group consisting of an antibody or fragmentthereof, polypeptide or fragments thereof, small molecules, and nucleicacids. In some embodiments, the agent comprises ACAT non-selectiveinhibitors, such as avasimibe or CI-976. In some embodiments, the agentcomprises avasimibe, CI-976, or both. In some embodiments, the effectiveamount of the agent is from about 0.01 mg/kg/day to about 200 mg/kg/day.In some embodiments, the agent is administered parenterally. Forexample, the agent may be administered subcutaneously, intravenously, orintraperitoneally. In some embodiments, the agent is administered by anintravenous injection. In one aspect, the present invention provides apharmaceutical composition comprising an agent inhibiting ACAT tosuppress food intake of a subject and one or more pharmaceuticallyacceptable excipient.

Suppressing food intake of a subject according to the methods describedherein, preferably result in a reduction of food intake of a subject byat least about 10% or greater relative to the amount, volume, weightand/or calorie of the food intake of the subject prior to treatment;more preferably, a reduction of food intake of a subject by at leastabout 20% or greater; more preferably, a reduction of food intake of asubject by at least about 30% or greater; more preferably, a reductionof food intake of a subject by at least about 40% or greater; morepreferably, a reduction of food intake of a subject by at least about50% or greater; even more preferably, a reduction of food intake of asubject by at least about 60% or greater; and most preferably, areduction of food intake of a subject by at least about 70% or greater.The food intake may be assessed by any reproducible means ofmeasurement. The food intake may be measured as an amount, a volume, aweight and/or a calorie.

In one aspect, the present invention provides methods of reducing bodyweight of a subject, comprising: administering an effective amount of anagent inhibiting ACAT to the subject and thereby suppressing food intakeof the subject. In some embodiments, the agent inhibits ACAT1 and/orACAT2. In some embodiments, the degree that the active agent inhibitsACAT1 may be similar to or less than the degree that the active agentinhibits ACAT 2. In some embodiments, the agent is selected from thegroup consisting of an antibody or fragment thereof, polypeptide orfragments thereof, small molecules, and nucleic acids. In someembodiments, the agent comprises ACAT non-selective inhibitors, such asavasimibe or CI-976. In some embodiments, the agent comprises avasimibeor CI-976. In some embodiments, the effective amount of the agent isfrom about 0.01 mg/kg/day to about 200 mg/kg/day. In some embodiments,the agent is administered parenterally. For example, the agent may beadministered subcutaneously, intravenously, or intraperitoneally. Insome embodiments, the agent is administered by an intravenous injection.In some embodiments, the agent suppresses a lipid accumulation in a bodyof the subject. In one aspect, the present invention provides apharmaceutical composition comprising an agent inhibiting ACAT tosuppress body weight of a subject and one or more pharmaceuticallyacceptable excipient.

In some embodiments, reducing body weight of a subject according to themethods described herein refers to suppressing gain of body weight of asubject. Suppressing gain of body weight of the subject preferablyresults in maintaining body weight of a subject within at most about110% or lesser relative to the body weight of the subject prior totreatment; more preferably, maintaining body weight of a subject withinat most about 109% or lesser; more preferably, maintaining body weightof a subject within at most about 108% or lesser; more preferably,maintaining body weight of a subject within at most about 107% orlesser; more preferably, maintaining body weight of a subject within atmost about 106% or lesser; more preferably, maintaining body weight of asubject within at most about 105% or lesser; more preferably,maintaining body weight of a subject within at most about 104% orlesser; more preferably, maintaining body weight of a subject within atmost about 103% or lesser; more preferably, maintaining body weight of asubject within at most about 102% or lesser; even more preferably,maintaining body weight of a subject within at most about 101% orlesser; and most preferably, maintaining body weight of a subject withinat most about 100%. The body weight may be assessed by any reproduciblemeans of measurement.

In some embodiments, reducing body weight of a subject according to themethods described herein refers to a reduction of body weight of asubject. A reduction of body weight of a subject preferably result in areduction of body weight of a subject by at least about 5% or greaterrelative to the body weight of the subject prior to treatment; morepreferably, a reduction of body weight of a subject by at least about10% or greater; more preferably, a reduction of body weight of a subjectby at least about 15% or greater; more preferably, a reduction of bodyweight of a subject by at least about 20% or greater; more preferably, areduction of body weight of a subject by at least about 25% or greater;more preferably, a reduction of body weight of a subject by at leastabout 30% or greater; even more preferably, a reduction of body weightof a subject by at least about 35% or greater; and most preferably, areduction of body weight of a subject by at least about 40% or greater.The body weight may be assessed by any reproducible means ofmeasurement.

In some embodiments, suppression of gaining body weight of a subjectaccording to the methods described herein is attributed to total WATweight loss. Total WAT weight of the subject is preferably decreased byat least about 5% or greater relative to the total WAT weight of thesubject prior to treatment; more preferably, decreased by at least about10% or greater; more preferably, decreased by at least about 15% orgreater; more preferably, decreased by at least about 20% or greater;more preferably, decreased by at least about 25% or greater; morepreferably, decreased by at least about 30% or greater; even morepreferably, decreased by at least about 35% or greater; and mostpreferably, decreased by at least about 40% or greater. The total WATweight may be assessed by any reproducible means of measurement.

Adipogenesis

Adipogenesis is the process of cell differentiation in whichpreadipocytes become mature adipocytes. There are four distinct stagesrequired for transition from preadipocytes to mature adipocytes: 1)growth arrest; 2) mitotic clonal expansion (additional 2-3 rounds ofcell cycle); 3) early differentiation; and 4) intermediate and terminaldifferentiation. Upon induction of adipogenesis, the growth-arrested3T3-L1 preadipocytes undergo mitotic clonal expansion withactivation/expression of cell cycle proteins, such as cdk2 and cyclinA.Mitotic clonal expansion is also accompanied by activation of cellularsignaling pathways, such as insulin-dependent phosphatidylinositol (PI)3-kinase and extracellular signal-regulated kinase (ERK) pathway, whichthen initiates the expression of a series of adipogenic transcriptionfactors. The major adipogenic transcription factors orchestrating theadipogenesis are CCAAT/enhancer binding protein (C/EBP)β, C/EBPα andperoxisome proliferator-activated receptorγ (PPARγ). C/EBPβ is expressedin the early stage of differentiation after clonal expansion, which isrequired for the subsequent expression of C/EBPα and PPARγ. During thetermination phase of adipogenesis, differentiating cells markedlyincrease expression of genes involved in de novo lipogenesis and TGsynthesis, including ATP citrate lyase, acetyl-CoA carboxylase (ACC),stearoyl-CoA desaturase 1 (SCD1), glycerol-3-phosphate acyltransferase(GPAT), glycerol-3-phosphate dehydrogenase, fatty acid synthase (FAS),glyceraldehyde-3-phosphate dehydrogenase (GAPDH), monoglycerideacyltransferase (MGAT), and diglyceride acyltransferase (DGAT),resulting in generation of new LDs and expansion of both adipocyte cellnumber and size. Given that adipocyte number is the major determinant ofincrease in adipose mass during childhood and adolescence both in leanand obese humans, inhibition of adipogenesis has been suggested to be aneffective strategy in lowering the generation and formation of newadipocytes and thereby decreasing or suppressing the overall adiposemass.

Adipogenesis has been reported to be associated with sharp increments inTG, CE and cholesterol in vitro. Additionally, approximately 280-foldincrease in cholesterol esterase activity is observed during 3T3-L1adipogenesis. During adipogenesis, most of the cellular cholesterol andCE in adipocytes reside in the plasma membrane and LDs. These reportsindicate a potential role of CE, a storage form of cholesterol, infunctional homeostasis of adipocytes.

Lipid Body

LDs, also referred to as lipid bodies, oil bodies or adiposomes, arelipid-rich cellular organelles that regulate the storage and hydrolysisof neutral lipids and are found largely in the adipose tissue. They alsoserve as a reservoir for cholesterol and acyl-glycerols for membraneformation and maintenance. LDs are found in all eukaryotic organisms andstore a large portion of lipids in mammalian adipocytes. Adipose tissueis largely viewed as an energy reservoir in a form of LD.

TGs are a major lipid found in LDs, and cholesterol and CE make up theneutral core of LDs found in adipocytes and macrophages, which arewrapped by a monolayer of phospholipids and other polar lipids. On theother hand, retinol esters are prominent neutral lipids found in LDs inliver stellate cells. The structure of LDs is maintained and stabilizedby various LD-binding proteins, such as patatin-like phospholipasedomain-containing proteins (e.g., perilipin1, perilipin2/adipophilin(ADRP), perilipin3/Tip47, and perilipin4/S3-12), cell death inducing DNAfragmentation factor proteins (CIDEs), fat-specific protein of 27 kDa(FSP27), and several lipases.

Defects in function of many of these LD-binding proteins have beenreported to be associated with impaired adipogenesis and LD generationin adipocytes. LD growth is facilitated by activated endoplasmicreticulum (ER) resident enzymes, such as DGAT for TG synthesis and ACATsfor cholesterol ester synthesis. These are also accompanied byactivation of de novo lipogenesis and fatty acid/cholesterol uptake inadipocytes. LDs are highly dynamic cellular compartments whosegeneration of fatty acids in response to an increased diet, anddegradation of fatty acids in response to a lack of systemic nutrientavailability are tightly controlled to maintain whole body energy andinflammation homeostasis.

Accordingly, alteration of intracellular LD accumulation is associatedwith a wide range of metabolic and inflammatory human diseases, such aslipodystrophy-associated insulin resistance, hepatic steatosis, andhypertension. Likewise, over-accumulation of TG- and CE-containing LDboth in adipose tissue and other non-adipose peripheral tissues, such asthe heart, liver and muscle, is also associated with neutral lipidstorage disease, cardiomyopathy, obesity, T2D, steatohepatitis, andcoronary heart disease.

Because TG is the predominant lipid in LD accumulated in most tissues,current therapies for treating the aforementioned chronic diseases havefocused on developing methods to suppress enzymes and genes involved infatty acid and TG synthesis. In addition to the role of TG in LD biologyin adipocytes, adipose tissue is also one of the largest reservoirs ofcholesterol and the size of cholesterol pool in adipocytes isproportional to the TG content and LD size. Despite the fact thatcholesterol content in adipocytes is known to be controlled bycholesterol synthesis, catabolism, efflux and/or influx, little is knownabout the role of cholesterol metabolism in LD growth and size inadipose tissue, and the development of adiposity.

ACATS/SOATS

Acyl-coenzyme A:cholesterol acyltransferase (ACAT), also known as sterolO-acyltransferase (SOAT), play an important role in cellular cholesterolesterification and thus late intestinal cholesterol absorption andhepatic lipoprotein secretion. ACATs catalyze the formation of CE fromlong chain fatty acid and cholesterol in the presence of ATP andcoenzyme A. The two substrates of this enzyme are acyl-CoA andcholesterol, whereas its two products are CoA and CE. This enzymebelongs to the family of transferases, specifically those ofacyltransferases transferring groups other than aminoacyl groups, themembrane-bound O-acyltransferases, and also participates in bile acidbiosynthesis.

Both ACAT1 and ACAT2 are located in the endoplasmic reticulum (ER)membrane and are both allosterically activated by cholesterol. WhileACAT1 is ubiquitously expressed in various tissues, ACAT2 is mainlyexpressed in the liver and intestine for supplying CE as the lipid coreof very low density lipoprotein and chylomicrons, respectively.

As CE constitutes most LDs in macrophages and has a positive correlationwith lipid-laden form cell formation, ACATs have been suggested to betherapeutic targets for atherosclerosis and hypercholesterolemia.Although ACAT2 knockout mice showed prevention against diet-inducedhypercholesterolemia by losing the cholesterol esterification activityin intestine and liver, the role of ACAT1 in atherosclerosis iscontroversial as hyperlipidemic mice with reconstituted ACAT1^(−/−)macrophages presented either detrimental or preventive function inatherosclerosis depending on the study designs.

In addition to the role of ACATs in hypercholesterolemia, CE synthesisappears to play important role in TG synthesis and LD biology. Forexample, yeast with a lack of TG and sterol ester displayed a defect inLD biogenesis with excess accumulation of neutral lipids in betweenmembrane bilayers. However, the role of CE and ACATs in adiposedevelopment and LD biogenesis in adipocytes has not yet been explored.

As mentioned above, obesity is characterized by increased LD due toexcess accumulation of lipids in adipose tissue. LD is composed ofmostly neutral lipids, such as triglycerides, cholesterol esters,phospholipids and LD-binding proteins. It is herein disclosed that ACAT1and ACAT2, are key enzymes involved in catalyzing the conversion of freecholesterol to CE, and are accordingly preventive and therapeutictargets for lowering fatty acid synthesis, LD accumulation and thedevelopment of obesity. In some embodiments, the use of avasimibe, acommercially available drug developed to control atherosclerosis is usedas an inhibitor of ACAT1 and ACAT2, and thereby regulating, treatingand/or preventing obesity.

Type-2 Diabetes (T2D)

Compositions and methods for treating T2D with improved blood glucosehomeostasis and reduction of body weight have been developed. Excessivewhite adipose tissue mass contributes to the development of obesity andinsulin resistance, which are attributed to increased adipocyte number(hyperplasia) and lipid storage (hypertrophy). Adipocyte hyperplasia andhypertrophy are governed by an increase in adipogenesis and LDaccumulation as described above. Therefore, regulating adipogenesisand/or LD generation is one of the effective strategies to controlinsulin resistance and obesity. As mentioned above, LD is composed ofmostly neutral lipids, such as triglycerides, cholesterol esters,phospholipids and LD-binding proteins. It is herein disclosed that ACAT1and ACAT2, the key enzymes catalyzing conversion of free cholesterol tocholesterol ester, are preventive and therapeutic target for modulatingglucose and insulin sensitivity and lowering body fat and food intake.

ACAT Inhibitors

The complete ACAT1 amino acid sequence can be found under GENBANK ®Accession No. AAH10942.1 (GI: 15012080) and is shown below (SEQ ID NO: 1):mavlpallrs garsrspllr rlvqeiryve rsyvskptlk evvivsatrt pigsflgslsllpatklgsi aiqgaiekag ipkeevkeay mgnvlqggeg qaptrqavlg aglpistpcttinkvcasgm kaimmasqsl mcghqikqet gslakicchv rrThe complete ACAT1 nucleic acid sequence can be found under GENBANK ®Accession No. BC010942 (GI: 15012079) and is shown below (SEQ ID NO: 2):ggggagtcta cgcctgtgga gccgatactc agcccactgc gaccatggct gtgctgccggcacttctgcg cagcggcgcc cgcagccgca gccccctgct ccggaggctg gtgcaggaaataagatatgt ggaacggagt tatgtatcaa aacccacttt gaaggaagtg gtcatagtaagtgctacaag aacacccatt ggatcttttt taggcagcct ttccttgctg ccagccactaagcttggttc cattgcaatt cagggagcca ttgaaaaggc agggattcca aaagaagaagtgaaagaagc atacatgggt aatgttctac aaggaggtga aggacaagct cctacaaggcaggcagtatt gggtgcaggc ttacctattt ctactccatg taccaccata aacaaagtttgtgcttcagg aatgaaagcc atcatgatgg cctctcaaag tcttatgtgt ggacatcagatcaagcaaga gacaggctcc ttagcaaaaa tatgctgtca tgtcaggagg tgagacctggacacacagaa gaatcaagat tctctcagat ctgagccctt catttttcag atgaagattttttttcagtg tgtctgagac agccacagag ttacagggct gagcatctgc catgtgacagtcattggaaa tagagtggtg aacaaaacat ttaaaaaaat ctgtacatgt gcaggtctctgttggaaaaa tgcctaaaag aaatgctgag tcaggatttg aacattttgg tatttgcaaatgctttccat aaaagttgta ccagttagac tttccaaaaa ttgtgtgact tgtctggatctgcaccacca ctgggtggta ccaaaccctt gtcaaactgg taggtgaaaa acggtcaccagatttagttt cagaactgtt tgtcatggaa agttttgtct taattgaagt attgtggttctctagcaaat gccatttgta ctatattgaa atactttcat ttaatattat tttattcatttgtggatata tacagtgact tataggcatt cttggaagtg ctttgttttg aatatttatgaccttagaaa acagtcagtt ttactttata atgaagaatt gataccttat tttctgtcacttattattgc catcaccccc agtaaaaagt acaagtgaat aaaacttaga tgagaactgattaagaattt ctctatttcg gaataggcaa aatatttatg tttctttggt atagagcttgcttgtctgta tgcctgatta aagactgtaa gaagatatta ttggctttat gtttacattaatgttttata ttaaactgtt tttaactagc gaaaaaaaaa aaaaaaaaaaThe complete ACAT2 amino acid sequence can be found under GENBANK ®Accession No. AAH00408.1 (GI: 12653279) and is shown below (SEQ ID NO: 3):mnagsdpvvi vsaartiigs fngalaavpv qdlgstvike vlkratvape dvsevifghvlaagcgqnpv rqasvgagip ysvpawscqm icgsglkavc lavqsigigd ssivvaggmenmskaphlay lrtgvkigem pltdsilcdg ltdafhnchm gitaenvakk wqvsredqdkvavlsqnrte naqkaghfdk eivpvlvstr rglievktde fprhgsniea msklkpyfltdgtgtvtpan asgindgaaa vvlmkksead krgltplari vswsqvgvep simgigpipaikqavtkagw sledvdifei neafaavsaa ivkelglnpe kvnieggaia lghplgasgcrilvtllhtl ermgrsrgva alcigggmgi amcvqreThe complete ACAT2 nucleic acid sequence can be found under GENBANK ®Accession No. BC000408.2 (GI: 38197144) and is shown below (SEQ ID NO: 4):ggagaagcaa gatgaatgca ggctcagatc ctgtggtcat cgtctcggcg gcgcggaccatcataggttc cttcaatggt gccttagctg ctgttcctgt ccaggacctg ggctccactgtcatcaaaga agtcttgaag agggccactg tggctccgga agatgtgtct gaggtcatctttggacatgt cttggcagca ggctgtgggc agaatcctgt tagacaagcc agtgtgggtgcaggaattcc ctactctgtt ccagcatgga gctgccagat gatctgtggg tcaggcctaaaagctgtgtg ccttgcagtc cagtcaatag ggataggaga ctccagcatt gtggttgcaggaggcatgga aaatatgagc aaggctcctc acttggctta cttgagaaca ggagtaaagataggtgagat gccactgact gacagtatac tctgtgatgg tcttacagat gcatttcacaactgtcatat gggtattaca gctgaaaatg tagccaaaaa atggcaagtg agtagagaagatcaggacaa ggttgcagtt ctgtcccaga acaggacaga gaatgcacag aaagctggccattttgacaa agagattgta ccagttttgg tgtcaactag aagaggtctt attgaagttaaaacagatga gtttcctcgc catgggagca acatagaagc catgtccaag ctaaagccttactttcttac tgatggaacg ggaacagtca ccccagccaa tgcttcagga ataaatgatggtgctgcagc tgtcgttctt atgaagaagt cagaagctga taaacgtgga cttacacctttagcacggat agtttcctgg tcccaagtgg gtgtggagcc ttccattatg ggaataggaccaattccagc cataaagcaa gctgttacaa aagcaggttg gtcactggaa gatgttgacatatttgaaat caatgaagcc tttgcagctg tctctgctgc aatagttaaa gaacttggattaaacccaga gaaggtcaat attgaaggag gggctatagc cttgggccac cctcttggagcatctggctg tcgaattctt gtgaccctgt tacacacact ggagagaatg ggcagaagtcgtggtgttgc agccctgtgc attgggggtg ggatgggaat agcaatgtgt gttcagagagaatgaattgc ttaaactttg aacaacctca atttcttttt aaactaataa agtactaggttgcaatatgt gaaatcagag gaccaaagta cagatggaaa ccatttccta catcacaaaaacccaagttt acagcttgta ctttacttta atgtgtaata ctcaactcaa ggtacaagacaattgcattt aacattgtta taaataaaag gaacatcaga tcaatcaaaa aaaaaaaaaa aaaThe mouse ACAT1 shRNA sequence is shown below (SEQ ID NO: 5):CCGGCCAACCAGAGACUAAACAUAUCUCGAGAUAUGUUUAGUCUCUGGUUGGUUUUUUGThe mouse ACAT2 shRNA sequence is shown below (SEQ ID NO: 6)CCGGUGCGGUGGUUCAUGAGUAUAUCUCGAGAUAUACUCAUGAACCACCGCAUUUUUUG

In one embodiment, the ACAT inhibitor is a small molecule. ExemplaryACAT small molecule inhibitors include avasimibe (CI-1011) (Pfizer);CI-976; CP113,818 (Pfizer); pactimibe; NTE-122 (Nissin Food ProductssCo., Ltd); F-1394 (Fujirebio Inc.); PD140296 (Parke-Davis); PD128042(Parke-Davis); PD132301-2 (Parke-Davis); octimibate; DuP128; 58-035;HL-004; SMP-500 (Sumitomo Pharmaceuticals Co.); CL-277,082; SKF-99085(Glaxo Smith-Kline); CS-505 (Sankyo Pharma); eflucimibe, F12511; E5324;FR145237 (Fujisawa Pharmaceutical Co., Ltd.); CL277,082; YM-17E,FR129169 (Fujisawa Pharmaceutical Co., Ltd.); and tamoxifen.

Small molecule inhibitors that inhibit both ACAT1 and ACAT2 are alsocontemplated. ACAT non-selective small molecule inhibitors that inhibitboth ACAT1 and ACAT2 may include, for example, avasimibe (CI-1011),CI-976 and pactimibe. Avasimibe is an oral ACAT non-selective inhibitorwith IC₅₀ of 10 μM for ACAT1 and IC₅₀ of 2.5 μM for ACAT2). Avasimibe isgenerally considered safe when administered to rats, dogs, and humans.Although avasimibe effectively lowered hypercholesterolemia, orallyadministrated avasimibe showed no effect on body weight, food intake andinsulin resistance.

In some embodiments, the small molecule is avasimibe,([[2,4,6-tris(1-methylethyl) phenyl]acetyl]-,2,6-bis(1-methylethyl)phenyl ester] sulfamic acid), a small moleculeoriginally developed to control atherosclerosis. In some embodiments,the small molecule is CI-976, (2,2-Dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide). CI-976 is an ACAT non-selective inhibitor with IC₅₀ of 5μM for ACAT1 and IC₅₀ of <1 μM for ACAT2.

In the present invention, ACAT small molecule inhibitors have an IC₅₀value in the range of 1 nM to 100 μM in vitro. When used in the methodsof this invention as described herein, one or more ACAT inhibitors areadministered to a subject (e.g., mammals, such as mice, rats, dogs,cats, horses, cows, and human) with the therapeutic benefit ofdecreasing, suppressing, reducing or ameliorating one or more signs orsymptoms of obesity and/or T2D including, but not limited to,accumulation of fatty acids, triglycerides and/or LDs in adipose tissueand/or liver; weight of adipose tissue and/or liver; blood glucose,insulin, free fatty acids, cholesterol, LDL, leptin and/or cytokines;whole body weight in the subjects as compared to subjects not receivingtreatment with ACAT inhibitor.

In some embodiments, the ACAT inhibitor is an antibody. The antibody ofthe present invention may be a polyclonal antisera or monoclonalantibody. The term antibody may include any of the various classes orsub-classes of immunoglobulin (e.g., IgG, IgA, IgM, IgD, or IgE derivedfrom any animal, e.g., any of the animals conventionally used, e.g.,sheep, rabbits, goats, or mice). Preferably, the antibody comprises amonoclonal antibody, e.g., an ACAT1 monoclonal antibody and/or an ACAT2monoclonal antibody.

An “isolated antibody,” as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds ACAT1 or that specifically binds ACAT2 and is substantially freeof antibodies that specifically bind antigens other than ACAT1 orACAT2). An isolated antibody that specifically binds ACAT1 or thatspecifically binds ACAT2 may, however, have cross-reactivity to otherantigens, such as ACAT1 or ACAT2 molecules from other species. Moreover,an isolated antibody may be substantially free of other cellularmaterial and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

An “antibody fragment” comprises a portion of an intact antibody,preferably the antigen binding and/or the variable region of the intactantibody. Examples of antibody fragments include Fab, Fab*, F(ab′)2 andFv fragments; diabodies; linear antibodies; single-chain antibodymolecules and multispecific antibodies formed from antibody fragments.Papain digestion of antibodies produced two identical antigen-bindingfragments, called “Fab” fragments, and a residual “Fc” fragment, adesignation reflecting the ability to crystallize readily. The Fabfragment consists of an entire L chain along with the variable regiondomain of the H chain (VH), and the first constant domain of one heavychain (CH1). Each Fab fragment is monovalent with respect to antigenbinding, i.e., it has a single antigen-binding site. Pepsin treatment ofan antibody yields a single large F(ab′)2 fragment, which roughlycorresponds to two disulfide linked Fab fragments having differentantigen-binding activity and is still capable of cross-linking antigen.Fab′ fragments differ from Fab fragments by having a few additionalresidues at the carboxy terminus of the CH1 domain including one or morecysteines from the antibody hinge region. Fab ′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear a free thiol group. F(ab′)2 antibody fragments originally wereproduced as pairs of Fab′ fragments which have hinge cysteines betweenthem. Other chemical couplings of antibody fragments are also known.

In some embodiments, exemplary ACAT1 antibodies may include antibodiespurchased from any suitable distributor, including, for example, Abcam,BD Biosciences, BioRad, Cell Signaling, EMD Milipore, Novus Biologicals,R&D Systems, and the like. For example, exemplary ACAT1 antibodies fromAbcam may include, but are not limited to Aviva Systems Biology, NovusBiologicals, LifeSpan BioSciences, GeneTex, Abnova Corporation, AtlasAntibodies, GenWay Biotech, Invigrogen Antibodies, Sigma-Aldrich, andBiovision.

In some embodiments, exemplary ACAT2 antibodies may include antibodiespurchased from any suitable distributor, including, for example, Abcam,BD Biosciences, BioRad, Cell Signaling, EMD Milipore, Novus Biologicals,R&D Systems, and the like. For example, exemplary ACAT2 antibodies fromAbcam may include, but are not limited to Aviva Systems Biology, NovusBiologicals, LifeSpan BioSciences, GeneTex, Abnova Corporation, AtlasAntibodies, GenWay Biotech, Invigrogen Antibodies, Sigma-Aldrich, andBiovision.

In some embodiments, the ACAT inhibitor is an inhibitory nucleic acid.As used herein, “inhibitory nucleic acid” is meant a double-strandedRNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimeticthereof, that when administered to a mammalian cell results in adecrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in theexpression of a target gene (e.g., ACAT1 and ACAT2). Typically, anucleic acid inhibitor comprises at least a portion of a target nucleicacid molecule, or an ortholog thereof, or comprises at least a portionof the complementary strand of a target nucleic acid molecule. Forexample, an inhibitory nucleic acid molecule comprises at least aportion of any or all of the nucleic acids delineated herein.

Inhibitory nucleic acid, siRNA may refer to a double stranded RNA.Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides inlength and has a 2 base overhang at its 3′ end. These dsRNAs can beintroduced to an individual cell or to a whole animal. For example, theymay be introduced systemically via the bloodstream. Such siRNAs are usedto downregulate mRNA levels or promoter activity.

In some embodiments, the ACAT inhibitor is an inhibitory nucleic acid,wherein the inhibitory nucleic acid is shRNA.

Methods

The methods of the invention include administration of ACAT inhibitors(e.g., an antibody or fragment thereof, peptides, polypeptides orfragments thereof, small molecules, and an inhibitory nucleic acid) forsuppressing food intake, reducing body weight; and treating orpreventing a metabolic disease.

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the compounds herein, such as a compound of theformulae herein to a subject in need thereof, including a mammal,particularly a human. Such treatment will be suitably administered tosubjects, particularly humans, suffering from, having, susceptible to,or at risk for a metabolic disease or symptom thereof. Determination ofthose subjects “at risk” can be made by any objective or subjectivedetermination by a diagnostic test or opinion of a subject or healthcare provider (e.g., genetic test, enzyme or protein marker, familyhistory, and the like).

The term “therapeutically effective amount” refers to an amount of anACAT inhibitor administered sufficient to suppress food intake, suppressbody weight; and treat, ameliorate a symptom of, reduce the severity of,or reduce the duration of metabolic diseases that are related toaccumulation of free fatty acids, TG and CE by the action of ACAT (e.g.,T2D and obesity). In some embodiments, the therapeutically effectiveamount is in an amount effective to achieve one or more of thefollowing: inhibit lipid droplet size accumulation in adipose tissue andinhibit enzymes that catalyze the conversion of free cholesterol tocholesterol ester (e.g., acyl-coenzyme A:cholesterol acyltransferase 1(ACAT1) and acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2).Additionally, the therapeutically effective amount may decrease orsuppress lipid accumulation (e.g., in adipose tissue), decrease orsuppress overall food intake, decrease or suppress blood glucose,decrease or suppress blood insulin, improves insulin sensitivity, anddecrease or suppress leptin. In some embodiments, the therapeuticallyeffective amount is in an amount effective to decrease, suppress, reduceor ameliorate one or more signs or symptoms of obesity including, butnot limited to, accumulation of fatty acids, triglycerides and/or LDs inadipose tissue and/or liver; weight of adipose tissue and/or liver;blood glucose, insulin, free fatty acids, cholesterol, LDL, leptinand/or cytokines; or whole body weight in the subjects as compared tosubjects not receiving treatment with ACAT inhibitor.

Dosages of the ACAT inhibitors may be varied depending upon therequirements of the patient. Utilizing the teachings provided herein, aneffective prophylactic or therapeutic treatment regimen can be plannedthat does not cause substantial toxicity and yet is effective to treatthe clinical symptoms demonstrated by the particular patient. The doseadministered to a patient should be sufficient to affect a beneficialtherapeutic response in the patient over time. The size of the dose alsowill be determined by the existence, nature, and extent of any adverseside-effects. Determination of the proper dosage for a particularsituation is within the skill of the art. This planning should involvethe careful choice of the ACAT inhibitor by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects. Generally, treatment isinitiated with smaller dosages which are less than the optimum dose ofthe ACAT inhibitor. Thereafter, the dosage is increased by smallincrements until the optimum effect under circumstances is reached.Dosage amounts and intervals can be adjusted individually to providelevels of the ACAT inhibitor effective for the particular clinicalindication being treated. This will provide a therapeutic regimen thatis commensurate with the severity of the individual's disease state.

The dosage and frequency (single or multiple doses) of the ACATinhibitors administered to a subject can vary depending upon a varietyof factors, for example, whether the mammal suffers from anotherdisease, and its route of administration; size, age, sex, health, bodyweight, body mass index, and diet of the recipient; nature and extent ofsymptoms of the disease being treated (e.g. symptoms and severity ofsuch symptoms), kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Dosages can beprovided in mg/kg/day units of measurement (which dose may be adjustedfor the patient's weight in kg, body surface area in m2, and age inyears). Other therapeutic regimens or agents can be used in conjunctionwith the methods and the ACAT inhibitors described herein. Adjustmentand manipulation of established dosages (e.g., frequency and duration)are well within the ability of those skilled in the art.

For any composition and the ACAT inhibitors described herein, thetherapeutically effective amount can be initially determined from cellculture assays. Target concentrations will be those concentrations ofthe ACAT inhibitors that are capable of achieving the methods describedherein, as measured using the methods described herein or known in theart. As is well known in the art, effective amounts of the ACATinhibitors for use in humans can also be determined from animal models.For example, a dose for humans can be formulated to achieve aconcentration that has been found to be effective in animals. The dosagein humans can be adjusted by monitoring effectiveness and adjusting thedosage upwards or downwards, as described above. Adjusting the dose toachieve maximal efficacy in humans based on the methods described aboveand other methods is well within the capabilities of the ordinarilyskilled artisan.

In some embodiments, an effective amount can range from about 0.001mg/kg to about 1000 mg/kg, more preferably 0.01 mg/kg to about 100mg/kg, more preferably 0.1 mg/kg to about 10 mg/kg; or any range inwhich the low end of the range is any amount between 0.001 mg/kg and 900mg/kg and the upper end of the range is any amount between 0.1 mg/kg and1000 mg/kg (e.g., 0.005 mg/kg and 200 mg/kg, 0.5 mg/kg and 20 mg/kg).Effective doses will also vary, as recognized by those skilled in theart, depending on the diseases treated, route of administration,excipient usage, and the possibility of co-usage with other therapeutictreatments such as use of other agents. In some embodiments, atherapeutically effective dose typically varies from 0.01 mg/kg/day toapproximately 750 mg/kg/day. In some embodiments, a therapeuticallyeffective dose is from about 0.01 mg/kg/day to about 200 mg/kg/day.

In some embodiments, the methods may comprise administering the ACATinhibitor according to a specified dosing schedule or therapeuticregimen. For example, the ACAT inhibitor can be administered once dailyor from two to five times daily. In some embodiments, the ACAT inhibitoris administered thrice daily, twice daily, once daily, fourteen days on(four times daily, thrice daily or twice daily, or once daily) and 7days off in a 3-week cycle, up to five or seven days on (four timesdaily, thrice daily or twice daily, or once daily) and 14-16 days off in3 week cycle, or once every two days, or once a week, or once every 2weeks, or once every 3 weeks. In some embodiments, the ACAT inhibitor isadministered for at least 25 days. In some embodiments, the ACATinhibitor is administered for at least 14 days. In some embodiments, theACAT inhibitor is administered for at least 8 days. In some embodiments,the ACAT inhibitor is administered for at least 6 days.

In some embodiments, the methods may comprise administering the ACATinhibitor systemically or locally by oral administration, subcutaneousinjection, intraperitoneal injection, intravenous injection, rectaladministration, topical application or inhalation.

Compositions

The present invention provides pharmaceutical compositions comprising aneffective amount of an ACAT inhibitor (e.g., an antibody or fragmentthereof, peptides, polypeptide or fragments thereof, small molecules,and inhibitory nucleic acids) and at least one pharmaceuticallyacceptable excipient or carrier, wherein the effective amount is asdescribed above in connection with the methods of the invention. Inaccordance with any of the embodiments described here, thepharmaceutical composition may be adapted for oral, buccal, orparenteral, subcutaneous, intraperitoneal, intravenous, rectal ortopical administration or inhalation. In some embodiments, thepharmaceutical composition is adapted for oral administration. In someembodiments, the pharmaceutical composition is adapted for parenteraladministration. In some embodiments, the pharmaceutical composition isadapted for intravenous injection. In some embodiments, the compositionmay comprise at least two ACAT inhibitors (e.g., avasimibe and CI-976)as the active agents.

A “pharmaceutical composition” is a formulation containing the compoundsdescribed herein in a pharmaceutically acceptable form suitable foradministration to a subject. The term “pharmaceutically acceptable”refers to those compounds, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Additionally, “pharmaceutically acceptable” means approved orapprovable by a regulatory agency of the Federal or a state governmentor the corresponding agency in countries other than the United States,or that is listed in the U.S. Pharmacopoeia or other generallyrecognized pharmacopoeia for use in animals, and more particularly, inhumans.

As used herein, “pharmaceutically acceptable excipient” means anexcipient that is useful in preparing a pharmaceutical composition thatis generally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. Examples of pharmaceuticallyacceptable excipients include, without limitation, sterile liquids,water, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), oils,detergents, suspending agents, carbohydrates (e.g., glucose, lactose,sucrose or dextran), antioxidants (e.g., ascorbic acid or glutathione),chelating agents, low molecular weight proteins, or suitable mixturesthereof.

As used herein “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The type of carrier can be selected basedupon the intended route of administration. Pharmaceutically acceptablecarriers include sterile aqueous solutions or dispersions and sterilepowders for the extemporaneous preparation of sterile topical solutionsor dispersion. The use of such media and agents for pharmaceuticallyactive substances is well known in the art. “Pharmaceutical carriers” or“carriers” as used herein can further include pharmaceuticallyacceptable carriers, excipients, or stabilizers which are nontoxic tothe cell or mammal being exposed thereto at the dosages andconcentrations employed. Often the physiologically acceptable carrier isan aqueous pH buffered solution. Examples of physiologically acceptablecarriers include buffers such as phosphate, citrate, and other organicacids; antioxidants including ascorbic acid; low molecular weight (lessthan about 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as Tween™, polyethylene glycol (PEG), and Pluronics™.

The pharmaceutical compositions can take any suitable form (e.g.,liquids, aerosols, solutions, inhalants, mists, sprays; or solids,powders, ointments, pastes, creams, lotions, gels, patches and the like)for administration by any desired route (e.g., pulmonary, inhalation,intranasal, oral, buccal, sublingual, parenteral, subcutaneous,intravenous, intramuscular, intraperitoneal, intrapleural, intrathecal,transdermal, transmucosal, rectal, and the like). The active therapeuticagent(s) may also be incorporated into microspheres, microcapsules,nanoparticles, liposomes, or the like for controlled release. Forexample, a pharmaceutical composition of the invention may be in theform of an aqueous solution or powder for aerosol administration byinhalation, in the form of a tablet or capsule for oral administration;in the form of a sterile aqueous solution or dispersion suitable foradministration by either direct injection or by addition to sterileinfusion fluids for intravenous infusion; or in the form of a lotion,cream, foam, patch, suspension, solution, or suppository for transdermalor transmucosal administration.

Oral formulations can include excipients as, for example, pharmaceuticalgrades of mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, cellulose, magnesium carbonate and the like. Thesecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. In someembodiments, oral pharmaceutical compositions will comprise an inertdiluent or assimilable edible carrier, or they may be enclosed in hardor soft shell gelatin capsule, or they may be compressed into tablets,or they may be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compounds may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltablets, troches, capsules, elixirs, suspensions, syrups, wafers, andthe like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be betweenabout 2 to about 75% of the weight of the unit, or preferably between25-60%. The amount of active compounds in such compositions is such thata suitable dosage can be obtained.

A pharmaceutical composition can be in the form of a sterile aqueoussolution or dispersion suitable for administration by either parenteraladministration or by addition to sterile infusion fluids for intravenousinfusion, and comprises a solvent or dispersion medium containing,water, ethanol, a polyol (e.g., glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, or one or morevegetable oils. Solutions or suspensions of the compound of the presentdisclosure as a free base or pharmacologically acceptable salt can beprepared in water suitably mixed with a surfactant. Examples of suitablesurfactants are given below.

The pharmaceutical composition may be administered parenterally byinjection, infusion or implantation (subcutaneous, intravenous,intramuscular, intraperitoneal, or the like) in dosage forms,formulations, or via suitable delivery devices or implants containingconventional, non¬toxic pharmaceutically acceptable carriers andadjuvants. The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intraarterial, intrasynovial, intrasternal, intrathecal, intralesionaland intracranial injection or infusion techniques. For parenteraladministration in an aqueous solution, for example, the solution shouldbe suitably buffered and the liquid diluent first rendered isotonic withsufficient saline or glucose. Sterile injectable solutions can beprepared by incorporating the active compounds in the required amount inthe appropriate solvent followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized activeingredients into a sterile vehicle which contains the basic dispersionmedium. Vacuum-drying and freeze-drying techniques, which yield a powderof the active ingredient plus any additional desired ingredients, can beused to prepare sterile powders for reconstitution of sterile injectablesolutions. Aqueous solutions, in particular, sterile aqueous media, areespecially suitable for intravenous, intramuscular, subcutaneous andintraperitoneal administration. These solutions are sterile andgenerally free of undesirable matter. These compositions may besterilized by conventional, well known sterilization techniques. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such asstabilizing, tonicity adjusting, pH adjusting and buffering agents,toxicity adjusting agents and the like, for example, sodium acetate,sodium chloride, potassium chloride, calcium chloride, sodium lactatehydrochloric acid, sodium hydroxide, 1,3-butanediol, Ringer's solution,isotonic sodium chloride solution, dextrose solution and the like. Theconcentration of active agent in these formulations can vary, and willbe selected primarily based on fluid volumes, viscosities, body weightand the like in accordance with the particular mode of administrationselected and the subject's needs. Solutions of the active compounds asfree base or pharmacologically acceptable salt can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations can contain a preservative (e.g.,methyl, ethyl or n-propyl p-hydroxybenzoate) to prevent the growth ofmicroorganisms. Such compositions are well known to those skilled in theart of pharmaceutical formulation. Formulations can be found inRemington: The Science and Practice of Pharmacy, supra. In cases whereone of the compounds is only sparingly or slightly soluble in water, adissolution enhancing or solubilizing agent can be added, or the solventmay include 10-60% w/w of propylene glycol or the like. The preparationof more, or highly, concentrated solutions for direct injection is alsocontemplated. DMSO can be used as solvent for extremely rapidpenetration, delivering high concentrations of the active agents to asmall area.

Pharmaceutical compositions can be delivered via intranasal or inhalablesolutions or sprays, aerosols or inhalants. Nasal solutions can beaqueous solutions designed to be administered to the nasal passages indrops or sprays. Nasal solutions can be prepared so that they aresimilar in many respects to nasal secretions. Thus, the aqueous nasalsolutions usually are isotonic and slightly buffered to maintain a pH of5.5 to 6.5. In addition, antimicrobial preservatives, similar to thoseused in ophthalmic preparations and appropriate drug stabilizers, ifrequired, may be included in the formulation. Various commercial nasalpreparations are known and can include, for example, antibiotics andantihistamines.

The pharmaceutical compositions for use in the methods of the presentdisclosure can further comprise one or more additives in addition to anycarrier or diluent (such as lactose or mannitol) that is present in theformulation. The one or more additives can comprise or consist of one ormore surfactants. Surfactants typically have one or more long aliphaticchains such as fatty acids which enables them to insert directly intothe lipid structures of cells to enhance drug penetration andabsorption. An empirical parameter commonly used to characterize therelative hydrophilicity and hydrophobicity of surfactants is thehydrophilic-lipophilic balance (“HLB” value). Surfactants with lower HLBvalues are more hydrophobic, and have greater solubility in oils, whilesurfactants with higher HLB values are more hydrophilic, and havegreater solubility in aqueous solutions. Thus, hydrophilic surfactantsare generally considered to be those compounds having an HLB valuegreater than about 10, and hydrophobic surfactants are generally thosehaving an HLB value less than about 10. However, these HLB values aremerely a guide since for many surfactants, the HLB values can differ byas much as about 8 HLB units, depending upon the empirical method chosento determine the HLB value.

Among the surfactants for use in the compositions of the disclosure arepolyethylene glycol (PEG)-fatty acids and PEG-fatty acid mono anddiesters, PEG glycerol esters, alcohol-oil transesterification products,polyglyceryl fatty acids, propylene glycol fatty acid esters, sterol andsterol derivatives, polyethylene glycol sorbitan fatty acid esters,polyethylene glycol alkyl ethers, sugar and its derivatives,polyethylene glycol alkyl phenols, polyoxyethylene-polyoxypropylene(POE-POP) block copolymers, sorbitan fatty acid esters, ionicsurfactants, fat-soluble vitamins and their salts, water-solublevitamins and their amphiphilic derivatives, amino acids and their salts,and organic acids and their esters and anhydrides.

The formulations of compounds can be presented in bulk or unit-dose(e.g., in single-dose ampoules) or multi-dose sealed containers in whicha suitable preservative may be added, such as ampules and vials. It isespecially advantageous to formulate pharmaceutical compositions indosage unit form for ease of administration and uniformity of dosage.Thus, the composition can be in unit dosage form. In such form thepreparation is subdivided into unit doses containing appropriatequantities of the active component. The compositions can be administeredin a variety of unit dosage forms depending upon the method ofadministration. The term “dosage unit form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved. A dosage unit forminclude, but are not limited to, an ampoule, a vial, a suppository, apowder, a tablet, a pill, a capsule, a lozenge, an IV bag, or a singlepump on an aerosol inhaler.

Combination Therapies

The present invention also provides methods comprising combinationtherapy. As used herein, the terms “combination therapy” or “co-therapy”include the administration of a therapeutically effective amount of anACAT inhibitor, with at least one additional active agent, as part of aspecific treatment regimen intended to provide a beneficial effect fromthe co-action of the active agents in the regimen. Thus, the inventionprovides methods of treating a subject for obesity or T2D using acombination therapy comprising an ACAT inhibitor and at least oneadditional active agent in a regimen for the treatment of the metabolicdisease (e.g., overweight, obesity, diabetes, T2D or related diseases).

The at least one additional active agent may be a therapeutic agent, forexample, an anti-inflammatory, or a non-therapeutic agent, andcombinations thereof. With respect to therapeutic agents, the beneficialeffect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutically active compounds.

Non-limiting examples of anti-inflammatory agents include21-acetoxypregnenolone, alclometasone, algestone, amcinonide,betamethosone diproprionate, budesonide, chloroprednisone, clobetasol,corticosterone, cortisone, cortivazol, deflazacort, desonide,dexamethasone alcohol, dexamethasone sodium phosphate, diflorasone,dutasteride, flumethasone pivalate, fluocinonide, fluorometholoneacetate, fluorometholone lcohol, fluticasone propionate, halcinonide,halobetasol propionate, halometasone, halopredone acetate,hydrocortamate, hydrocortisone, hydroflumethiazide lotoprendoletabonate, medrysone, prednisolone acetate, prednisolone sodiumphosphate, rimexolone, hydrocortisone, hydrocortisone acetate,lodoxamide tromethamine, difluprednate, or a combination thereof. In oneaspect, the steroidal anti-inflammatory agent may be a corticosteroiddrug, such as prednisolone acetate.

The anti-inflammatory agents may be used in any suitable amounts. Forexample, in some embodiments, such anti-inflammatory agents may be in aconcentration of from about 0.01% to 10.0% by weight. Theanti-inflammatory agents can be present at about 0.01, 0.1, 0.2, 0.3,0.4, 0.5, 1.0, 2.0, 5.0, and 10 percent by weight or any amount inbetween these amounts. In some embodiments, the anti-inflammatory may bein a concentration of about 0.05% (w/v) to about 1.0% (w/v).

A “combined synergistic amount” as used herein refers to the sum of afirst amount (e.g., an amount of an ACAT inhibitor) and a second amount(e.g., an amount of an anti-inflammatory agent) that results in asynergistic effect (i.e., an effect greater than an additive effect).Therefore, the terms “synergy”, “synergism”, “synergistic”, “combinedsynergistic amount”, and “synergistic therapeutic effect” which are usedherein interchangeably, refer to a measured effect of compoundsadministered in combination where the measured effect is greater thanthe sum of the individual effects of each of the compounds administeredalone as a single agent.

A “combined additive amount” as used herein refers to the sum of a firstamount (e.g., an amount of an ACAT inhibitor) and a second amount (e.g.,an amount of an anti-inflammatory agent) that results in an additiveeffect (i.e., an effect equal to the sum of the effects). Therefore, theterms “additive”, “combined additive amount”, and “additive therapeuticeffect” which are used herein interchangeably, refer to a measuredeffect of compounds administered in combination where the measuredeffect is equal to the sum of the individual effects of each of thecompounds administered alone as a single agent.

Combinations of agents or compositions can be administered eitherconcomitantly (e.g., as a mixture), separately but simultaneously (e.g.,via separate intravenous lines) or sequentially (e.g., one agent isadministered first followed by administration of the second agent).Thus, the term combination is used to refer to concomitant, simultaneousor sequential administration of two or more agents or compositions. Thecourse of treatment is best determined on an individual basis dependingon the particular characteristics of the subject and the type oftreatment selected. The treatment, such as those disclosed herein, canbe administered to the subject on a daily, twice daily, bi-weekly,monthly or any applicable basis that is therapeutically effective. Thetreatment can be administered alone or in combination with any othertreatment disclosed herein or known in the art. The additional treatmentcan be administered simultaneously with the first treatment, at adifferent time, or on an entirely different therapeutic schedule (e.g.,the first treatment can be daily, while the additional treatment isweekly).

The combined administrations contemplates co-administration, usingseparate formulations or a single pharmaceutical formulation, andconsecutive administration in either order, wherein preferably there isa time period while both (or all) active agents simultaneously exerttheir biological activities.

EXAMPLES

Embodiments herein are further illustrated by the following examples anddetailed protocols. However, the examples are merely intended toillustrate embodiments and are not to be construed to limit the scopeherein. The contents of all references and published patents and patentapplications cited throughout this application are hereby incorporatedby reference.

Example 1: A Positive Correlation of ACAT1 Expression with Adipogenesisand Obesity

To examine the function of ACATs in obesity, the expression patterns ofACAT1 and ACAT2 genes, and their gene products during adipogenesis ofmurine 3T3-L1 preadipocytes in vitro were examined. ACAT1 mRNA level wasmarkedly increased in adipocytes from 2 days after initiation ofadipogenesis (i.e., D2) as judged by real-time PCR assay (FIG. 1 ).However, ACAT2 mRNA level was similar between preadipocytes (D0) andmature adipocytes (D6) while a temporal reduction of ACAT2 level wasobserved at D2 (FIG. 1 ). In addition, white adipose tissue (WAT)isolated from high fat diet-induced obese mice displayed elevated mRNAlevel of ACAT1 and reduced mRNA level of ACAT2 when compared with thosein lean mice as judged by real-time PCR assay. Leptin level was measuredin WAT from lean and obese mice to ensure the development of obesity(FIG. 2 ). In addition, brown adipose tissue (BAT) from obese miceexhibited elevated levels of both ACAT1 and ACAT2. Uncoupling protein-1(UCP-1) level was measured in BAT from lean and obese mice as aBAT-specific marker protein (FIG. 2 ). However, liver from lean andobese mice exhibited similar levels of ACAT1 and ACAT2 (FIG. 2 ).

Example 2: Inhibition of Lipid Accumulation in Adipocytes by ACATInhibitors (Avasimibe and CI-976)

To determine the effect of ACAT inhibitors on lipid accumulation inadipocytes, murine 3T3-L1 preadipocytes were differentiated to matureadipocytes in the presence or absence of avasimibe (0 μM, 1 μM, 5 μM, 10μM and 20 μM) for 6 days. Avasimibe solution was prepared by dissolvingin dimethyl sulfoxide (DMSO). At day 6, preadipocytes andavasimibe-treated differentiated adipocytes were subjected to Oil Red Ostaining to visualize accumulated intracellular lipids. Oil Red Ostained lipids were then extracted for spectrometric quantification oflipids. Intracellular lipids were decreased in a dose-dependent mannerby up to 60% (FIG. 3A and FIG. 3B). Avasimibe-inhibited lipidaccumulation was confirmed by Coherent Anti-Stokes Raman Scattering(CARS) microscopy, which is a dye-free non-invasive imaging techniquefor visualization of lipid-containing molecules in biological systems(FIG. 4 ). Similar with avasimibe, treatment of differentiatingadipocytes with CI-976, a non-selective ACAT inhibitor, for 6 days inthe concentration range of 0 μM, 5 μM, 15 μM and 20 μM also resulted ina decrease in lipid accumulation as judged by Oil Red O staining (FIG.5A and FIG. 5B). Thus, these data indicate that avasimibe and CI-976were effective in markedly reducing or suppressing lipid dropletaccumulation in differentiating adipocytes in vitro.

To characterize the effect of avasimibe treatment on mRNA levels ofgenes involved in lipid metabolism and adipocyte function,differentiated 3T3-L1 preadipocytes in the presence or absence ofavasimibe (20 μM) for 6 days were subjected to real-time PCR assay forthe analysis of expression of genes involved in synthesis of fatty acidsand TGs such as PPARγ, sterol regulatory element binding protein 1c(SREBP1c), MGAT1, DGAT2, FAS and SCD-1. As shown in FIG. 6 , avasimibetreatment resulted in a marked decrease in mRNA levels of genes involvedin lipid synthesis in adipocytes.

Example 3: Avasimibe Lowers Lipid Accumulation in Adipocytes ThroughModulation of SREBP1 Expression and Function on Lipid Synthesis

Because SREBP1 plays a key role in de novo fatty acids and TG synthesisand the data in FIG. 6 show a potential inhibitory role of avasimibe insuppression of SREBP1-downstream genes involved in fatty acid and TGsynthesis, the ability of avasimibe treatment to alter SREBP1 proteinexpression and de novo fatty acid synthesis in adipocytes was tested.Avasimibe treatment resulted in elevated levels of the 125 kDa inactiveprecursor form of SREBP1 in adipocytes as judged by Western blotanalysis using antibodies specific to SREBP1 and f-actin (a proteinloading control) (FIG. 7 ).

Using deuterium labeled glucose-d7, the effect of avasimibe on de novofatty acid synthesis in adipocytes treated with or without avasimibe wasexamined. Glucose-d7 incorporated fatty acids were visualized bystimulated Raman scattering (SRS) microscopy. SRS imaging showed reducedlevel of LD accumulation (i.e., vibrational image of C-H bond-rich lipidmolecules) and the signal intensity of glucose-d7 accumulated in the LDsas a result of replacement of all the hydrogen atoms in fatty acids byglucose-derived deuterium atoms (i.e., vibrational image of C-Dbond-rich lipid molecules) in avasimibe-treated adipocytes (FIG. 8 ).These results indicate an inhibitory or suppressive role of avasimibe inde novo fatty acid synthesis potentially through inhibition of SREBP1function.

Example 4: Effect of Avasimibe on Cholesterol Metabolism in Adipocytes

Previous studies reported an adipogenesis-dependent increase incholesterol accumulation in adipocytes. Similarly, adipogenesis wasassociated with elevated level free cholesterol with little change in CElevel. Moreover, avasimibe treatment resulted in a significantly reducedlevel of cholesterol and a complete inhibition of CE accumulation inadipocytes during adipogenesis (FIG. 9 ). This was accompanied by amarked decrease in mRNA levels of genes involved in cholesterol uptakesuch as scavenger receptor class B member 1 (SR-BI) and CD-36 inadipocytes treated with avasimibe when compared with those in controladipocytes (FIG. 10 ). Using a25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol(25-NBD cholesterol)-based ACAT assay, avasimibe indeed inhibited theACAT activity, thereby lowering accumulation of fluorescence-labeled CEin adipocytes (FIG. 11 ). Collectively, the results indicate thatavasimibe not only inhibits synthesis of fatty acids and TGs, but alsoinhibits cholesterol uptake and accumulation of free cholesterol and CEin adipocytes, possibly through suppression of expression of genesinvolved in cholesterol uptake from the medium.

Example 5: Effect of shRNA Inhibition of ACAT1 on Lipid DropletAccumulation in Adipocytes

The direct role of ACAT1 on synthesis of fatty acids and TGs inadipocytes by lentivirus-mediated ACAT1 knockdown using its specificshRNA was tested. FIG. 12 shows a gene knockdown efficiency of the shRNAto ACAT1 with no influence on ACAT2 expression level in adipocytes.Similar with the effect of avasimibe on adipocytes, ACAT1 knockdownresulted in suppression of lipid accumulation in adipocytes up to 40%than control ShRNA adipocytes as judged by Oil Red O staining (FIG. 13Aand FIG. 13B), and a marked decrease in mRNA levels of genes involved inlipid synthesis as indicated by real-time PCR assay (FIG. 14 ).Similarly, ACAT2 knockdown resulted in suppression of lipid accumulationin adipocytes up to 55% than control ShRNA adipocytes as judged by OilRed O staining (FIG. 15A and FIG. 15B). ACAT2 knocking down resulted ina dramatic reduction in mRNA levels of genes involved in TG synthesis(FIG. 16 ). Here, ACAT inhibitors, avasimibe and CI-976, have beenidentified to be highly effective for anti-obesity treatment in a mousemodel of obesity. The cell-based studies indicated that ACAT inhibitionand ACAT1 knockdown reduced or suppressed synthesis of fatty acid, TGand lipid droplets in adipocytes with lower level of accumulation offree cholesterol and CE. The cell-based studies also suggest that theinhibitory or suppressive role of ACAT inhibition and ACAT1 knockdown inlipid synthesis in adipocytes is largely due to suppression of SREBP1expression and function, and expression of its-downstream genes involvedin synthesis of fatty acids and TG.

Example 6: Anti-Diabetic Effect of Avasimibe in Mice with High-FatDiet-Induced Obesity an Insulin Resistance

Tissue- and isoform-specific regulation of ACATs has been proposed to betherapeutic strategy for cholesterol dysregulated diseases, such asatherosclerosis. However, the therapeutic role of ACATs in obesity hasnever been explored. Using high fat diet-fed C57BL/6J obese and diabeticmice, a daily i.p. injection of avasimibe (20 mg/kg body weight) forabout 2-week resulted in about 25% reduction or suppression in totalbody weight gain (FIG. 17A and FIG. 17B) along with a significant lossof whole body fat mass (FIG. 18A) and white adipose tissue (Ing:inguinal adipose tissue, Epi: epididymal adipose tissue, Retro:retroperitoneal adipose tissue) weight (FIG. 18B) with no noticeableliver toxicity as judged by alanine transaminase (ALT) assay (FIG. 19 ).Avasimibe treatment resulted in approximately 50% decrease in foodintake (FIG. 20A). This in turn reduced energy expenditure (FIG. 20B)with a shift of energy utilization pattern of tested mice from dietarycarbohydrates to lipids stored in adipose tissue, as demonstrated byreduced respiratory exchange ratio in avasimibe-treated mice and bloodglucose levels (FIG. 20C) compared with control mice. Moreover, a 2-weekof avasimibe administration to obese and diabetic mice resulted inmarked reduction of blood glucose (FIG. 21A) and insulin levels (FIG.21B), improved glucose tolerance (FIG. 21C) and homeostatic modelassessment of insulin resistance (HOMA-IR) values (FIG. 21D).

Avasimibe-treated mice exhibited significantly improved blood lipidprofile such as a decrease in free cholesterol (FIG. 22A), CE (FIG.22B), and TG (FIG. 22C). Consistent with reduced or suppressed adiposetissue mass shown in FIG. 18A and FIG. 18B, circulating leptin level, anadipose tissue-secreted satiety hormone that regulates whole body energybalance by inhibiting food intake behavior, was markedly suppressed byavasimibe treatment (FIG. 22D).

Since ACAT1 inhibitor resulted in a dramatic decrease in food intake,the reason for avasimibe's action to decrease or suppress food intakewas tested for its efficacy on lowering obesity phenotypes and insulinresistance. To do this, mice were pair-fed with the amount of foodconsumed by avasimibe-administrated mice for 8 days. Bothavasimibe-treated mice and pair-fed mice showed similar levels of bodyweight loss (FIG. 23A) and food intake (FIG. 23B) with little change inthe fecal energy content (FIG. 23C and FIG. 23D).

Interestingly, mice in pair-fed group showed lower blood glucose levelcompared with the control group but didn't reach to the level found fromavasimibe-treated mice (FIG. 23E). Thus, these results provided evidencethat inhibition of ACAT1 activity in a diet-induced obese and diabeticmice are a useful strategy for treating T2D with a benefit of bodyweight loss.

Here, ACAT inhibitors, avasimibe and CI-976, have been identified as ahighly effective anti-diabetes treatment. It is anticipated that themethod of the present invention could involve administration of an ACAT1inhibitor either alone or in combination with other drugs by any desiredroute known to have efficacy in treating symptoms of T2D and obesity.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

Example 7: Proteomic Analysis of Proteins Influenced by Avasimibe inAdipocytes Using LC MS/MS

In order to get a comprehensive view about the role of ACAT in the LDdevelopment during adipogenesis and explore new molecular targetsinfluenced by ACAT inhibition, we employed a global proteomic approachto investigate the effect of ACAT inhibition on adipocyte proteomes in a3T3-L1 preadipocyte model.

Material and Methods

1. Cell Preparation and Digestion

3T3-L1 preadipocytes were induced to differentiate into matureadipocytes with the same culture condition as described elsewhere. Thecells were treated with avasimibe (10 μM) or DMSO during day 2 to 6,which is the period when LD forms. Avasimibe or DMSO was replacedfreshly every 48 h. Each condition was cultured in triplicate. On day 6,adipocytes were washed with 1×PBS and scraped off the culture dishes.Adipocyte cell pellets were collected after centrifugation andsubsequently digested with trypsin (Sigma-Aldrich, St. Louis Mo., USA)at the ratio of 1:50 (μg/pg) trypsin to protein.

2. LC MS/MS and Proteomic Analysis

The tryptic peptides after trypsin digestion were separated on a nano LCsystem (1100 Series LC, Agilent Technologies, Santa Clara, Calif.) asdescribed previously (D'Aquila, et al. 2015). Briefly, peptides wereconcentrated with Agilent 300SB-C18 enrichment column and separated withC18 reversed phase ZORBAX 300SB-C18 column. High resolution hybrid iontrap mass spectrometer LTQ-Orbitrap LX (Thermo Fisher Scientific,Waltham Mass., USA) was employed to identify the peptides in thedata-dependent positive acquisition mode. These experiments were done inBindley Bioscience Center. We used the MaxQuant computational proteomicsplatform version 1.5.3.17 (Cox and Mann, 2008) to analyze the resultsfrom LC MS/MS analysis. The Mus musculus (24754 entries) sequence fromUNIPROT retrieved on Sep. 1, 2015 and a common contaminants databasewere used as our reference databases. MaxQuant was set with parametersas follows: minimum peptides length was set to seven amino acids, and“Match between runs” interval was set to one minute; The FASTA databaseswere randomized and 1% of protein false discovery rate was allowed; Withtrypsin digestion, two missed cleavages were accepted; Threemodifications per peptide were allowed, including one fixed modificationas “Iodoethanol” and variable modifications as “Oxidation (M)” and“Acetyl (Protein N-term)”; Initial precursor was set to 0.07 andfragment mass tolerance set to 0.02 Da; Data were analyzed with“Label-free quantification” (LFQ) checked.

The MaxQuant results were used in in-house script to remove all thecommon contaminant proteins and to calculate the average LFQ intensityvalues for the biological replicates and log transform [log 2(x)]. Themissing values were left blank unless indicated. A two tailed studentt-test was performed on the LFQ intensity.

Results and Discussion

1. Proteins Identified Through LC MS/MS

The proteomes of 3T3-L1 adipocytes (D6) cultured with or withoutavasimibe (10 μM) (n=3) during the LD formation stage (i.e., Day 2-Day6) were analyzed. 669 proteins were detected and identified. Theanalysis was limited to proteins which were identified in all threebiological replicates (217+10+9=236 proteins). Further analysis (FIG. 24) revealed that ACAT inhibition decreased or suppressed the levels of 76identified proteins, increased the levels of 102 identified proteins,and did not alter the levels of 58 proteins significantly.

When the data were filtered with the criteria that an abundance changeof >2-fold increase or decrease (log 2 T−log 2 C>1 or <−1, and emptyvalues were filled with half of the lowest intensity value, i.e.,6.3462−1=5.3462), it was found that avasimibe treatment increased thelevels of 22 proteins and decreased the levels of 53 proteins. These 75proteins were further analyzed by the Database for Annotation,Visualization and Integrated Discovery (DAVID) protein analysis, and theresults listed in FIG. 25 -FIG. 27 are described below.

2. Proteomic Result is Consistent with the Previous Finding In Vitro.

This proteomics analysis found that 7 identified proteins associatedwith FA metabolic process were downregulated with avasimibe treatment.These 7 proteins were categorized into 2 groups: 1) lipogenesis relatedproteins: Acyl-CoA synthetase long-chain family member 1 (86%reduction), FAS (only identified in the control group); 2) mitochondrialFA β-oxidation related proteins: Acyl-CoA dehydrogenase (56% reduction),enoyl CoA hydratase short chain 1 (64% reduction), hydroxyacyl-CoAdehydrogenase (64% reduction), acetyl-CoA acyltransferase 1 (61%reduction), carnitine O-acetyltransferase (only identified in thecontrol group) and hydroxysteroid (17-β) dehydrogenase 4 (38%reduction).

Additionally, it was found that ACAT inhibition also downregulated theprotein levels of adipocyte markers, including adiponectin (84%reduction) and fatty acid binding protein 4 (64% reduction). There were3 identified proteins associated with cholesterol metabolism. Amongthem, cytochrome B5 Reductase 3 (involved in cholesterol biosynthesis)and superoxide dismutase 1 (associated with negative regulation ofcholesterol biosynthesis) were not altered significantly by ACATinhibition, while sterol carrier protein 2 (for bile acid biosynthesis)was downregulated by 36%. Considering de novo cholesterol synthesis islimited in adipocytes, it was speculated that ACAT inhibition may notalter cholesterol biosynthesis significantly, although enzyme activityassay is needed to confirm it.

Notably, avasimibe is known to inhibit cytochrome P450 (CYP450), themajor drug metabolizing enzymes (Sahi, et al. 2004). Similarly, thecurrent proteomics result suggested that ACAT inhibition reduced orsuppressed the protein level of cytochrome B5 Type A (67% reduction),which is involved in CYP450 pathway. Taken together, the proteomeanalysis support that ACAT inhibition downregulated proteins involved inlipogenesis and delayed adipogenesis in vitro.

3. Cytoskeleton May be Involved in the Suppression of LD Development byACAT Inhibition.

During adipogenesis, cell morphology changes from flat to spherical, andthis morphological change is explained by cytoskeletal remodeling,including fragmentation of microtubules and depolymerization of actinmicrofilaments during adipogenesis (Smas and Sul 1995; Welsh, et al.2004). Moreover, it is well known that almost all the detectedcytoskeletal protein levels decrease during adipogenesis (Soukas, et al.2001; Welsh, et al. 2004). The Database for Annotation, Visualizationand Integrated Discovery (DAVID) analysis of the aforementionedproteomics study revealed that the proteins upregulated by avasimibewere primarily associated with cytoskeletons. This could explain howACAT inhibition delayed adipogenesis process. It is known thatcytoskeleton proteins are required for stabilizing the newly generatedLDs in human adipocytes (Heid, et al. 2014). However, it is unclearwhether avasimibe suppressed LD development through cytoskeletonremodeling or not. These results propose a hypothesis whether ACATsaffect LD development via cytoskeleton remodeling.

1. A method of controlling the amount of food intake of an animal, themethod comprising parenterally administering at least once in a month tothe animal in need thereof a therapeutically effective amount of acomposition comprising as an active agent an acyl-coenzyme A:cholesterolacyltransferase (ACAT) inhibitor or a pharmacologically acceptable saltthereof.
 2. The method of claim 1, wherein the active agent isincorporated into a microsphere, a microcapsule, a nanoparticle, or aliposome.
 3. The method of claim 1, wherein the composition isformulated in the form of an applicator stick, a lotion, a cream, anointment, a gel, a jelly, a paint, a powder, an aerosol, a foam, apatch, a suspension, or a solution.
 4. The method of claim 1, whereinthe ACAT inhibitor non-selectively inhibits in vivo both ACAT1 andACAT2.
 5. The method of claim 4, wherein the degree that the ACATinhibitor inhibits ACAT1 is less than the degree that the ACAT inhibitorinhibits ACAT2.
 6. The method of claim 1, wherein the ACAT inhibitor isselected from the group consisting of: avasimibe (CI-1011); CI-976(PD128042); pactimibe (CS-505); NTE-122; F-1394; PD140296; PD132301-2;octimibate; DuP128; Sandoz 58-035; HL-004; SMP-500; CL-277,082;SKF-99085; eflucimibe (F12511); E5324; FR145237; YM-17E; FR129169; andtamoxifen.
 7. The method of claim 1, wherein the composition isadministered subcutaneously, intravenously, intraperitoneally,intranasally, or intracerebrally.
 8. The method of claim 1, wherein thecomposition further comprises an additional active agent.
 9. The methodof claim 8, wherein the additional active agent is an anti-obesityagent.
 10. The method of claim 9, wherein the anti-obesity drug isselected from the group consisting of Sibutramine, Phentermine,Mazindol, Diethylpropion, Leptin, Orlistat, a Beta-3 agonist, andRimonabant.
 11. The method of claim 1, wherein an additional compositioncomprising an additional active agent is co-administered simultaneouslyor separately.
 12. The method of claim 1, wherein the administration isby an injection.
 13. The method of claim 1, wherein the pharmaceuticalcomposition is administered to the animal at least once in a week. 14.The method of claim 1, wherein the composition is administered at leastonce in a day.
 15. The method of claim 1, wherein the animal is a canineor feline.
 16. The method of claim 1, wherein the active agent reducesbody weight of the animal.
 17. The method of claim 1, wherein the activeagent improves a diabetic condition of the animal.